Compositions for selection of aptamers

ABSTRACT

The present disclosure describes compositions and methods for rapid selection of both binding and functional oligonucleotides (DNA, RNA, or any natural or synthetic analog of these). In certain embodiments, provided herein are flow cells (e.g., flow cells for an Illumina sequencing instrument or a Polonator sequencing instrument) comprising within its flow chamber a plurality of immobilized aptamer clusters (e.g., from an aptamer library described herein) and, optionally, one or more target cells (e.g., cancer cells, immune cells, etc.) and/or a detectable indicator of cellular function (e.g., a fluorescent indicator of apoptosis, cell proliferation, gene or protein expression, etc.). In certain embodiments, provided herein are methods of using such an aptamer cluster-containing flow cell to identify functional aptamers from an aptamer library (e.g., in a sequencing instrument, such as an Illumina sequencing instrument).

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/599,970, filed Oct. 11, 2019, which is a divisional of U.S. patentapplication Ser. No. 16/165,267, filed Oct. 19, 2018, now U.S. patentSer. No. 10/501,743, which is a continuation of International PatentApplication No. PCT/IB2018/000418, filed Mar. 30, 2018, which claims thebenefit of priority to U.S. Provisional Patent Application Ser. No.62/478,993, filed Mar. 30, 2017, each of which is hereby incorporated byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 30, 2018, isnamed ANB-001_25_SL.txt and is 2,065 bytes in size.

BACKGROUND

Aptamers are short, single-stranded nucleic acid oligomers that can bindto a specific target molecule. Aptamers are typically selected from alarge random pool of oligonucleotides in an iterative process. Morerecently, aptamers have been successfully selected in cells, in-vivo andin-vitro.

The selection of aptamers, their structure-function relationship, andtheir mechanisms of action are all poorly-understood. Although more than100 aptamer structures have been solved and reported, almost norecurring structural motifs have been identified.

A variety of different aptamer selection processes have been describedfor identifying aptamers capable of binding to a particular target.However, the ability to rapidly and conveniently identify aptamers ableto mediate a desirable functional effect on a target of interest wouldhave a profound impact on aptamer therapeutics.

SUMMARY

Provided herein are compositions and methods related to theidentification of aptamers that bind to and/or mediate a functionaleffect on a target (e.g., a target cell or a target molecule). Forexample, in certain embodiments, provided herein are flow cells (e.g.,flow cells for an Illumina sequencing instrument or a Polonatorsequencing instrument) comprising within its flow chamber a plurality ofimmobilized aptamer clusters (e.g., from an aptamer library describedherein) and, optionally, one or more target cells (e.g., cancer cells,immune cells, etc.) and/or a detectable indicator of cellular function(e.g., a fluorescent indicator of apoptosis, cell proliferation, gene orprotein expression, etc.). In certain embodiments, provided herein aremethods of using such an aptamer cluster-containing flow cell toidentify functional aptamers from an aptamer library (e.g., in asequencing instrument, such as an Illumina sequencing instrument).

In certain aspects, provided herein are methods for identifying one ormore aptamers that specifically bind to a target (e.g., a target cell, atarget virus, a target protein, a topographic feature on a cell). Insome embodiments, the methods comprise (i) contacting a plurality ofaptamer clusters immobilized on a surface (e.g., a flow cell surface)with the target; and (ii) identifying immobilized aptamer clusters thatbind to the target. In certain embodiments, the methods further compriseperforming a wash step after step (i) to remove unbound target fromsurface (e.g., a flow cell surface). In some embodiments, the target isdetectably labeled (e.g., fluorescently labeled).

In some aspects, provided herein are methods for identifying one or moreaptamers that modulate a property of a cell (e.g., a prokaryotic cell ora eukaryotic cell). In some embodiments, the methods comprise (i)contacting a plurality of aptamer clusters immobilized on a surface withthe cell; and (ii) identifying the immobilized aptamer clusters thatmodulate the property of the cell (e.g., cell viability, cellproliferation, gene expression, cell morphology, etc.). In someembodiments, the methods further comprise performing a wash step afterstep (i) to remove unbound target from surface (e.g., a flow cellsurface). In some embodiments, the cell comprises a detectable label(e.g., a fluorescent dye, such as a calcium sensitive dye, a cell tracerdye, a lipophilic dye, a cell proliferation dye, a cell cycle dye, ametabolite sensitive dye, a pH sensitive dye, a membrane potentialsensitive dye, a mitochondrial membrane potential sensitive dye, or aredox potential dye). In some embodiments, a change in the property ofthe cell causes a change in the properties of the detectable label whichare detected in order to identify the immobilized aptamer clusters thatmodulate the property of the cell.

In some aspects, provided herein are methods for identifying one or moreaptamers that possess a functional property (e.g., an enzymaticproperty) that modulates a target (e.g., a target molecule, such as atarget protein). In some embodiments, the methods comprise (i)contacting a plurality of aptamer clusters immobilized on a surface withthe target; and (ii) identifying the immobilized aptamer clusters thatmodulate the target (e.g., that cleave the target, that induce achemical or structural change on the target, etc.). In some embodiments,the methods further comprise performing a wash step after step (i) toremove unbound target from flow cell.

In certain embodiments, the methods further comprise the generation ofthe immobilized aptamer clusters. In some embodiments, the immobilizedaptamer clusters are generated by: (a) immobilizing a plurality ofaptamers (e.g., from an aptamer library) on the surface; and (b)amplifying the plurality of immobilized aptamers locally on the flowcell surface (e.g., via bridge PCR amplification or rolling circleamplification) to form the plurality of immobilized aptamer clusters. Insome embodiments, the methods further comprise removing thecomplementary strands from the immobilized aptamer clusters to providesingle stranded immobilized aptamer clusters. In certain embodiments,the immobilized aptamer clusters are sequenced following step (b) (e.g.,using Illumina sequencing or Polonator sequencing). In some embodiments,the immobilized aptamer clusters are generated by printing aptamerclusters (e.g., from an aptamer library) directly on the surface. Insome embodiments, the methods comprise the generation of the aptamerlibrary (e.g., through chemical nucleic acid synthesis).

In certain aspects, provided herein are compositions comprising aptamerclusters (e.g., a clustered aptamer library). In certain embodiments,the aptamer clusters are immobilized on a solid support (e.g., a flowcell). In certain embodiments, the composition further comprises atarget (e.g., a target cell, a target molecule, a target protein). Incertain embodiments, the composition further comprises a detectablelabel (e.g., a fluorescent dye, such as a calcium sensitive dye, a celltracer dye, a lipophilic dye, a cell proliferation dye, a cell cycledye, a metabolite sensitive dye, a pH sensitive dye, a membranepotential sensitive dye, a mitochondrial membrane potential sensitivedye, or a redox potential dye). In some embodiments the compositioncomprises 10⁴-10⁹ aptamer clusters (e.g., at least about 10⁴, 5×10⁴,10⁵, 5×10⁵, 10⁶, 5×10⁵, 10⁶, 5×10⁶, 10⁷, 5×10⁷, or 10⁸ aptamer clusters.In some embodiments, each cluster in the library contains 10³-10⁶) ofaptamers (e.g., at least about 10³, 5×10³, 10⁴, 5×10⁴, 10⁵, 5×10⁵aptamers per cluster.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 is a schematic diagram of aptamer library synthesis, sequencingand target identification work flow according to certain embodimentsdescribed herein.

FIG. 2 is a bar graph showing the binding of target cells (Hana cells)to a library of aptamers (Lib), short or long aptamers of randomsequence, aptamer outputs of SELEX selection process for the specifictarget cells cycles 6 and 7 (Cyc6 and Cyc7 respectively), specificaptamer sequences from SELEX selection process (Apt1 and Apt2), and anempty lane (empty) on a Illumina GAIIx flow-cell. Cells were run downflow cell lanes, and bound cells counted (bound vs. unbound, expressedas fraction, 1=100% of cells).

FIG. 3 is an image of a cell bound to aptamers on a flow cell. The imageshows the movement of the cell relative to the surface over time. Theimage shows that the cell is retained by the immobilized aptamercluster, rather than attached to the surface itself, and is thus free tomove but confined to that location. Imaging was performed on an IlluminaGAIIx.

FIG. 4 is a schematic representation of certain aptamer structuresaccording to certain exemplary embodiments provided herein.

FIG. 5 includes three panels and illustrates an exemplary aptameridentification method according to certain embodiments disclosed herein.Panel A, is a schematic representation of a flow-cell based aptamerdetection method. An initial aptamer library (SEQ ID NOS 5-7,respectively, in order of appearance) is sequenced and a flow-cell isgenerated in which aptamers from the library are clustered at a uniquecoordinates. Positive targets (e.g. tumor biopsy cells) and negativetargets (e.g. peripheral bloods) are labeled with fluorescent indicatorsfor one or more biological effects (e.g. apoptosis) and introduced intodifferent lanes of the flow-cell on which the aptamer clusters areimmobilized. Following an incubation, fluorescence is detected andassociated with a position on the flow cell on which an aptamer clusteris immobilized. Aptamers are scored based on effectiveness yes/no(Effect/No Effect) and selectivity yes/no (Selective/Not Selective). Thehighest scored oligos (E+S) are synthesized and validated. Panel B showsa photograph of a flow-cell following sequencing, held inside acustom-built adapter for a screening fluorescent microscope. Panel C isa screenshot of a fluorescent microscope image obtained duringperformance of an embodiment of the claimed method showing target cellsfollowing incubation, with apoptotic cells producing a positive signal.

DETAILED DESCRIPTION General

Provided herein are methods and composition related to theidentification of aptamers that bind to and/or mediate a functionaleffect on a target (e.g., a target cell or a target molecule). Incertain embodiments the methods comprise contacting the target to aplurality of aptamer clusters immobilized on a surface. Thus, in someembodiments, the method comprises flowing a solution comprising thetarget across the surface of a flow cell to which clusters of aptamershave been immobilized and detecting which aptamer clusters bind to,interact with and/or mediate a functional effect on the target.

In certain embodiments, the sequence of each immobilized aptamer clusteris known and/or determined, for example, by sequencing the aptamerclusters or by printing aptamers of known sequences onto predeterminedpositions of the surface. Thus, by determining the position on thesurface at which the target binds to, interacts with and/or is modulatedby an aptamer cluster, the relevant effect can be associated with theaptamer sequence at that position.

For example, in some embodiments, aptamers that bind to a target areidentified by running a composition comprising a target that comprises adetectable label (e.g., a fluorescent label) across a surface to whichaptamer clusters of known sequences are immobilized at known positions.The positions on the surface at which the target is retained aredetermined (e.g., using fluorescent microscopy), indicating that theaptamers immobilized at those positions bind to the target.

In certain embodiments, aptamers that functionally modulate a target areidentified by running a composition comprising a target that comprises adetectable label indicative of the function being modulated (e.g., afluorescent dye, such as a calcium sensitive dye, a cell tracer dye, alipophilic dye, a cell proliferation dye, a cell cycle dye, a metabolitesensitive dye, a pH sensitive dye, a membrane potential sensitive dye, amitochondrial membrane potential sensitive dye, or a redox potentialdye) across a surface to which aptamer clusters of known sequences areimmobilized at known positions. The positions on the surface at whichthe detectable label indicates that the target is modulated aredetermined (e.g., using fluorescent microscopy), indicating that theaptamers immobilized at those positions are able to modulate the target.

In certain aspects, also provided herein are methods and compositionsrelated to the creation of immobilized of aptamer clusters on a surface.In some embodiments, aptamers (e.g., from an aptamer library disclosedherein) are immobilized onto a surface, such as a flow cell surface. Insome embodiments, a localized amplification process, such as bridgeamplification or rolling circle amplification, is then performed togenerate aptamer clusters. The aptamer clusters can then be sequenced(e.g., by Illumina sequencing or Polonator sequencing) in order toassociate the sequence of each aptamer cluster with a position on thesurface. The complementary strands can be stripped in order to generatesingle-stranded aptamer clusters. The surface (e.g., flow cell) is thenready for use in an aptamer identification method provided herein.

Conveniently, in certain embodiments, all the steps in the methodsprovided herein can be performed in an Illumina sequencing instrument,such as an Illumina GAIIx instrument.

In certain aspects, provided herein are compositions comprising aptamerclusters (e.g., a clustered aptamer library generated during theperformance of a method provided herein). In certain embodiments, theaptamer clusters are immobilized on a solid support (e.g., a flow cell).In certain embodiments, the composition further comprises a target(e.g., a target cell, a target molecule, a target protein). In certainembodiments, the composition further comprises a detectable label (e.g.,a fluorescent dye, such as a calcium sensitive dye, a cell tracer dye, alipophilic dye, a cell proliferation dye, a cell cycle dye, a metabolitesensitive dye, a pH sensitive dye, a membrane potential sensitive dye, amitochondrial membrane potential sensitive dye, or a redox potentialdye). In some embodiments the composition comprises 10⁴-10⁹ aptamerclusters (e.g., at least about 10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁵, 10⁶,5×10⁶, 10⁷, 5×10⁷, or 10⁸ aptamer clusters. In some embodiments, eachcluster in the library contains 10³-10⁶) of aptamers (e.g., at leastabout 10³, 5×10³, 10⁴, 5×10⁴, 10⁵, 5×10⁵ aptamers per cluster.

In some embodiments, the target can be a cell of any type (e.g.prokaryotic cell, such as a bacterium, or a eukaryotic cell, such as amammalian cell), a virus, a protein, and/or a particle. In someembodiments, the particle is attached to the target.

In some embodiments, the detectable label is a fluorescent reporter offunction. In some embodiments the fluorescent reporter of function is acell death reporter, a redox potential reporter, a membrane integrityreporter. In some embodiments, the fluorescent reporter of function is avirus reporter, such as a capsid integrity reporter (e.g., a reporterfor measuring the capsid integrity and or functions of a virus). In someembodiments, the fluorescent reporter of function is a protein reporter,such as a protein integrity reporter (i.e., a reporter for measuring aprotein's structural integrity and stability) or a protein denaturationreporter (i.e., a reporter to detect protein denaturation).

Examples of cell death reporters are 7-AAD, and Annexin V fluorophore.In certain embodiments, the target is linked to, bound by or comprises adetectable label that allows for the detection of a biological orchemical effect on the target. In some embodiments, the detectable labelis a fluorescent dye. Non-limiting examples of fluorescent dyes include,but are not limited to, a calcium sensitive dye, a cell tracer dye, alipophilic dye, a cell proliferation dye, a cell cycle dye, a metabolitesensitive dye, a pH sensitive dye, a membrane potential sensitive dye, amitochondrial membrane potential sensitive dye, and a redox potentialdye. In one embodiment, the target is labeled with a calcium sensitivedye, a cell tracer dye, a lipophilic dye, a cell proliferation dye, acell cycle dye, a metabolite sensitive dye, a pH sensitive dye, amembrane potential sensitive dye, a mitochondrial membrane potentialsensitive dye, or a redox potential dye.

In certain embodiments, the target is labeled with an activationassociated marker, an oxidative stress reporter, an angiogenesis marker,an apoptosis marker, an autophagy marker, a cell viability marker, or amarker for ion concentrations. In yet another embodiment, the target islabeled with an activation associated marker, an oxidative stressreporter, an angiogenesis marker, an apoptosis marker, an autophagymarker, a cell viability marker, or a marker for ion concentrationsprior to exposure of aptamers to the target.

In some embodiments, the target cell is labeled with a calcium sensitivedye, a cell tracer dye, a lipophilic dye, a cell proliferation dye, acell cycle dye, a metabolite sensitive dye, a pH sensitive dye, amembrane potential sensitive dye, a mitochondrial membrane potentialsensitive dye, or a redox potential dye. In certain embodiments, thetarget cell is labeled with an activation associated marker, anoxidative stress reporter, an angiogenesis marker, an apoptosis marker,an autophagy marker, a cell viability marker, or a marker for ionconcentrations. In yet another embodiment, target cell is labeled withan activation associated marker, an oxidative stress reporter, anangiogenesis marker, an apoptosis marker, an autophagy marker, a cellviability marker, or a marker for ion concentrations prior to exposureof aptamers to the cell. In some embodiments, the target cell is labeledafter to exposure of aptamers to the target. In one embodiment, thetarget cell is labeled with a fluorescently-labeled antibody orantigen-binding fragment thereof, annexin V, a fluorescently-labeledfusion protein, a fluorescently-labeled sugar, or fluorescently labeledlectin. In one embodiment, the target cell is labeled with afluorescently-labeled antibody or antigen-binding fragment thereof,annexin V, a fluorescently-labeled fusion protein, afluorescently-labeled sugar, or fluorescently labeled lectin afterexposure of aptamers to the cell.

In some embodiments the aptamer clusters are immobilized on a flow cell(e.g., a flow cell is used for image-based DNA sequencing, such as,Illumina instrument flow cell and/or Polonator sequencer flow cell). Incertain embodiments, the flow cell can be made of any material. In someembodiments the flow cell is made of plastic, glass, polymer, or metal.In some embodiments, the flow cell is a plate, a tray, or a chip. Insome embodiments, the flow cell contains between its floor and ceilingone or more of the following: air, water, aqueous buffer, culturemedium, serum, patient-derived sample (e.g. blood, plasma, serum,urine), matrix (e.g. gel), a polymer, and/or a protein (e.g. collage, orpatient-derived extracellular matrix). In some embodiments, the flowcell contains targets (e.g., target cells). In some embodiments, theflow cell (e.g. its floor and/or its ceiling) is coated with a blocker,such as, a polymer, a protein, an oligo, a lipid, and/or a chemicalgroup. In some embodiments, the flow cell contains an anchor of anylength to bind the target at proximity to clusters. In some embodiments,the anchor is a polymer, a protein, an oligo, a lipid, and/or a chemicalgroup.

In some embodiments, the aptamer clusters are arranged randomly in theflow cell. In other embodiments, the aptamer clusters are arrangedaccording to a specific pattern in the flow cell. In some embodiments,the flow cell is fixed at any stage. In some embodiments, thecomposition further comprises a fixative. In some embodiments, the flowcell is used for iterative process of oligo selection. In otherembodiments, the flow cell is used in non-iterative process of oligoselection.

In certain embodiments, the target is labeled with an activationassociated marker, an oxidative stress reporter, an angiogenesis marker,an apoptosis marker, an autophagy marker, a cell viability marker, or amarker for ion concentrations. In yet another embodiment, the target islabeled with an activation associated marker, an oxidative stressreporter, an angiogenesis marker, an apoptosis marker, an autophagymarker, a cell viability marker, or a marker for ion concentrationsprior to exposure of aptamers to the target.

Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The articles “a” and “an” are used herein to refer to one or to morethan one (e.g., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the term “aptamer” refers to a short (e.g., less than200 bases), single stranded nucleic acid molecule (ssDNA and/or ssRNA)able to specifically bind to a protein or peptide target or to atopographic feature on a target cell.

As used herein, the term “aptamer cluster” refers to a collection oflocally immobilized aptamers (e.g., at least 10) of identical sequence.

The term “binding” or “interacting” refers to an association, which maybe a stable association, between two molecules, e.g., between an aptamerand target, e.g., due to, for example, electrostatic, hydrophobic, ionicand/or hydrogen-bond interactions under physiological conditions.

As used herein, two nucleic acid sequences “complement” one another orare “complementary” to one another if they base pair one another at eachposition.

As used herein, two nucleic acid sequences “correspond” to one anotherif they are both complementary to the same nucleic acid sequence.

The term “modulation”, when used in reference to a functional propertyor biological activity or process (e.g., enzyme activity or receptorbinding), refers to the capacity to either up regulate (e.g., activateor stimulate), down regulate (e.g., inhibit or suppress) or otherwisechange a quality of such property, activity, or process. In certaininstances, such regulation may be contingent on the occurrence of aspecific event, such as activation of a signal transduction pathway,and/or may be manifest only in particular cell types.

As used herein, “specific binding” refers to the ability of an aptamerto bind to a predetermined target. Typically, an aptamer specificallybinds to its target with an affinity corresponding to a K_(D) of about10⁻⁷ M or less, about 10⁻⁸ M or less, or about 10⁻⁹ M or less and bindsto the target with a K_(D) that is significantly less (e.g., at least 2fold less, at least 5 fold less, at least 10 fold less, at least 50 foldless, at least 100 fold less, at least 500 fold less, or at least 1000fold less) than its affinity for binding to a non-specific and unrelatedtarget (e.g., BSA, casein, or an unrelated cell, such as an HEK 293 cellor an E. coli cell).

As used herein, the Tm or melting temperature of two oligonucleotides isthe temperature at which 50% of the oligonucleotide/targets are boundand 50% of the oligonucleotide target molecules are not bound. Tm valuesof two oligonucleotides are oligonucleotide concentration dependent andare affected by the concentration of monovalent, divalent cations in areaction mixture. Tm can be determined empirically or calculated usingthe nearest neighbor formula, as described in Santa Lucia, J. PNAS (USA)95:1460-1465 (1998), which is hereby incorporated by reference.

The terms “polynucleotide” and “nucleic acid” are used hereininterchangeably. They refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Polynucleotides may have any three-dimensional structure, andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: coding or non-coding regionsof a gene or gene fragment, loci (locus) defined from linkage analysis,exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA,ribozymes, cDNA, synthetic polynucleotides, recombinant polynucleotides,branched polynucleotides, plasmids, vectors, isolated DNA of anysequence, isolated RNA of any sequence, nucleic acid probes, andprimers. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modified,such as by conjugation with a labeling component.

Aptamer Libraries

In certain embodiments, the methods and compositions provided hereinrelate to the identification of aptamers having desired properties fromamong the aptamers present in an aptamer library. As used herein, anaptamer library is a collection of nucleic acid molecules (e.g., DNAand/or RNA) having distinct sequences (e.g., at least 10², 10³, 10⁴,10⁵, 10⁶ or 10⁷ distinct sequences) and wherein at least a subset of thenucleic acid molecules is structured such that they are capable ofspecifically binding to a target protein, peptide, or cellulartopographic feature. In some embodiments, any library of potentialaptamers can be used in the methods and compositions provided herein.

In some embodiments, the aptamer library used in the methods andcompositions provided herein comprises, consists of and/or consistsessentially of nucleic acid molecules (e.g., DNA and/or RNA) having asequence according to Formula (I):

P1-R-P2  (I),

wherein P1 is a 5′ primer site sequence of about 10 to 100 bases inlength, about 10 to 50 bases in length, about 10 to 30 bases in length,about 15 to 50 bases in length or about 15 to 30 bases in length; P2 isa 3′ primer site sequence of about 10 to 100 bases in length, about 10to 50 bases in length, about 10 to 30 bases in length, about 15 to 50bases in length or about 15 to 30 bases in length; and R is a sequencecomprising randomly positioned bases of about at least 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 bases in length and/or nomore than about 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60,55 or 50 bases in length.

In one embodiment, R is a sequence comprising about 25% A. In anotherembodiment, R is a sequence comprising about 25% T. In anotherembodiment, R is a sequence comprising about 25% G. In anotherembodiment, R is a sequence comprising about 25% C. In yet anotherembodiment, R is a sequence comprising about 25% A, about 25% T, about25% G, and about 25% C.

In some embodiments, the aptamer library used in the methods andcompositions provided herein comprises, consists of and/or consistsessentially of nucleic acid molecules (DNA and/or RNA) having a sequenceaccording to Formula (I):

P1-R″-P2  (I),

wherein P1 is a 5′ primer site sequence of about 10 to 100 bases inlength, about 10 to 50 bases in length, about 10 to 30 bases in length,about 15 to 50 bases in length or about 15 to 30 bases in length; P2 isa 3′ primer site sequence of about 10 to 100 bases in length, about 10to 50 bases in length, about 10 to 30 bases in length, about 15 to 50bases in length or about 15 to 30 bases in length; and R″ is a sequenceof about at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75or 80 bases in length and/or no more than about 120, 115, 110, 105, 100,95, 90, 85, 80, 75, 70, 65, 60, 55 or 50 bases in length comprisingrandomly positioned bases from a biased mixture or any combination ofrandom strings with repetitive or biased strings.

In some embodiments, the aptamer library used in the methods andcompositions provided herein comprises, consists of and/or consistsessentially of nucleic acid molecules (DNA and/or RNA) having a sequenceaccording to Formula II (an exemplary schematic representation isprovided in FIG. 4A),

P1-S1-L1-S1*-S2-L2-S2*-P2  (II),

wherein:

P1 is a 5′ primer site sequence of about 10 to 100 bases in length,about 10 to 50 bases in length, about 10 to 30 bases in length, about 15to 50 bases in length or about 15 to 30 bases in length; P2 is a 3′primer site sequence of about 10 to 100 bases in length, about 10 to 50bases in length, about 10 to 30 bases in length, about 15 to 50 bases inlength or about 15 to 30 bases in length; S1 and S2 are eachindependently a stem region sequence of at least one base (e.g., ofabout 4 to 40 bases in length or 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39 or 40 bases in length); S1* is acomplementary sequence to S1; S2* is a complementary sequence to S2; L1and L2 are each independently a Loop region sequence of at least onebase (e.g., of about 1 to 50 bases in length or 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49 or 50 bases in length); and S1-L1-S1*-S2-L2-S2* iscollectively about at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75 or 80 bases in length and/or no more than about 120, 115,110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55 or 50 bases in length.

In some embodiments, the aptamer library used in the methods andcompositions provided herein comprises, consists of and/or consistsessentially of nucleic acid molecules (DNA and/or RNA) having a sequenceaccording Formula III (an exemplary schematic representation is providedin FIG. 4B):

P1-S1-L1-S2-L2-S2*-L1-S1*-P2  (III),

wherein:

P1 is a 5′ primer site sequence of about 10 to 100 bases in length,about 10 to 50 bases in length, about 10 to 30 bases in length, about 15to 50 bases in length or about 15 to 30 bases in length; P2 is a 3′primer site sequence of about 10 to 100 bases in length, about 10 to 50bases in length, about 10 to 30 bases in length, about 15 to 50 bases inlength or about 15 to 30 bases in length;

S1 and S2 are each independently a stem region sequence of at least onebase (e.g., of about 4 to 40 bases in length or 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 bases in length); S1*is a complementary sequence to S1; S2* is a complementary sequence toS2;

L1 and L2 are each independently a Loop region sequence of at least onebase (e.g., of about 1 to 50 bases in length or 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49 or 50 bases in length); and

S1-L1-S2-L2-S2*-L1-S1* is collectively about at least 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 bases in length and/or nomore than about 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60,55 or 50 bases in length.

In some embodiments, the aptamer library used in the methods andcompositions provided herein comprises, consists of and/or consistsessentially of nucleic acid molecules (DNA and/or RNA) having a sequenceaccording Formula IV (an exemplary schematic representation is providedin FIG. 4C):

P1-Lib-M1/M2-D-M1/M2*-Lib-P2  (IV),

wherein:

P1 is a 5′ primer site sequence of about 10 to 100 bases in length,about 10 to 50 bases in length, about 10 to 30 bases in length, about 15to 50 bases in length or about 15 to 30 bases in length; P2 is a 3′primer site sequence of about 10 to 100 bases in length, about 10 to 50bases in length, about 10 to 30 bases in length, about 15 to 50 bases inlength or about 15 to 30 bases in length;

Lib is sequence having a formula selected from: (i) R; (ii) R″; (iii)S1-L1-S1*-S2-L2-S2*; and (iv) S1-L1-S2-L2-S2*-L1-S1*;

R is a sequence comprising randomly positioned bases of about at least10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 bases inlength and/or no more than about 120, 115, 110, 105, 100, 95, 90, 85,80, 75, 70, 65, 60, 55 or 50 bases in length;

R″ is a sequence of about at least 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75 or 80 bases in length and/or no more than about 120,115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55 or 50 bases inlength comprising randomly positioned bases from a biased mixture or anycombination of random strings with repetitive or biased strings; 51 andS2 are each independently a stem region sequence of at least one base(e.g., of about 4 to 40 bases in length or 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 bases in length); S1* is acomplementary sequence to 51; S2* is a complementary sequence to S2;

L1 and L2 are each independently a Loop region sequence of at least onebase (e.g., of about 1 to 50 bases in length or 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49 or 50 bases in length);

wherein S1-L1-S1*-S2-L2-S2* is collectively about at least 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 bases in length and/orno more than about 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65,60, 55 or 50 bases in length;

D is a spacer sequence comprising at least one base (e.g., of about 1 to20 bases in length or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or 20 bases in length); M1 is a multimer-forming domainsequence of about 10 to 18 bases in length or 10, 11, 12, 13, 14, 15,16, 17 or 18 bases in length that enables a strand of the sequence tointeract with another strand that contains a complementary domain; and

M2 is a complementary domain of M1 comprising a strand that interactswith a strand of the M1 sequence.

In some embodiments, the aptamer library used in the methods andcompositions provided herein comprises, consists of and/or consistsessentially of nucleic acid molecules (DNA and/or RNA) having a sequenceaccording Formula V (an exemplary schematic representation is providedin FIG. 4D):

P1-Lib-T*-Lib-P2  (V),

wherein:

P1 is a 5′ primer site sequence of about 10 to 100 bases in length,about 10 to 50 bases in length, about 10 to 30 bases in length, about 15to 50 bases in length or about 15 to 30 bases in length; P2 is a 3′primer site sequence of about 10 to 100 bases in length, about 10 to 50bases in length, about 10 to 30 bases in length, about 15 to 50 bases inlength or about 15 to 30 bases in length;

Lib is sequence having a formula selected from: (i) R; (ii) R″; (iii)S1-L1-S1*-S2-L2-S2*; and (iv) S1-L1-S2-L2-S2*-L1-S1*;

R is a sequence comprising randomly positioned bases of about at least10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 bases inlength and/or no more than about 120, 115, 110, 105, 100, 95, 90, 85,80, 75, 70, 65, 60, 55 or 50 bases in length;

R″ is a sequence of about at least 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75 or 80 bases in length and/or no more than about 120,115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55 or 50 bases inlength comprising randomly positioned bases from a biased mixture or anycombination of random strings with repetitive or biased strings;

S1 and S2 are each independently a stem region sequence of at least onebase (e.g., of about 4 to 40 bases in length or 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 bases in length); S1*is a complementary sequence to S1; S2* is a complementary sequence toS2;

L1 and L2 are each independently a Loop region sequence of at least onebase (e.g., of about 1 to 50 bases in length or 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49 or 50 bases in length);

wherein S1-L1-S1*-S2-L2-S2* is collectively about at least 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 bases in length and/orno more than about 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65,60, 55 or 50 bases in length;

T is a second strand bound by Watson/Crick or Hoogsteen base pairing toany part of the Lib sequence or T*, wherein the strand optionallycontains unpaired domains on its 5′ and 3′ ends (e.g., to facilitateattachment of a functional moiety to the aptamer); and

T* is a dedicated domain sequence (e.g., of about 4 to 40 bases inlength or 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39 or 40 bases in length).

In some embodiments of the Formulae above, R is randomly positionedbases from any random mixture (e.g., for canonical bases, 25% A, 25% T,25% G, 25% C) of about at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75 or 80 bases in length and/or no more than about 120, 115,110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55 or 50 bases in length.

In one embodiment of the Formulae above, R is a sequence comprisingabout 25% A. In another embodiment, R is a sequence comprising about 25%T. In another embodiment, R is a sequence comprising about 25% G. Inanother embodiment, R is a sequence comprising about 25% C. In yetanother embodiment, R is a sequence comprising about 25% A, about 25% T,about 25% G, and about 25% C.

In some embodiments of the Formulae above, R″ is a sequence comprisingcomprises randomly positioned bases from a biased mixture (e.g., forcanonical bases, any mixture deviating from 25% per base). In someembodiments, R″ is a sequence that comprises about 0%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% A. In someembodiments, R″ is a sequence that comprises about 0%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% T. In someembodiments, R″ is a sequence that comprises about 0%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% C. In someembodiments, R″ is a sequence that comprises about 0%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% G. In someembodiments, R″ is a sequence that comprises any combination of randomstrings (string is any sequence including a single base) with repetitiveor biased strings.

In some embodiments of the Formulae above, R″ is randomly positionedbases from a biased mixture (e.g., for canonical bases, any mixturedeviating from 25% per base); or any combination of random strings(string is any sequence including a single base) with repetitive orbiased strings of about at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75 or 80 bases in length and/or no more than about 120, 115,110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55 or 50 bases in length.

In some embodiments of the Formulae above, S1 is a stem region sequenceof at least 1 base or more. In other embodiments, S1 is a stem regionsequence of between about 4 to 40 bases in length or 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 bases in length.

In some embodiments of the Formulae above, S2 is a stem region sequenceof at least 1 base or more. In other embodiments, S2 is a stem regionsequence of between about 4 to 40 bases in length or 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 bases in length.

In some embodiments of the Formulae above, L1 is a Loop region sequenceof at least one base. In other embodiments, L1 is a Loop region sequenceof about 1 to 50 bases in length or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49 or 50 bases in length.

In some embodiments of the Formulae above, L2 is a Loop region sequenceof at least one base. In other embodiments, L2 is a Loop region sequenceof about 1 to 50 bases in length or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49 or 50 bases in length.

In some embodiments of the Formulae above, T may include unpaireddomains on its 5′ and 3′ ends, or it may be a padlock tail (e.g., a loopbetween two domains paired with the library).

The aptamers of the present disclosure may contain any number of stemsand loops, and other structures comprised of stems and loops (e.g.,hairpins, bulges, etc.). In some embodiments, the loops in the aptamercontain bases implanted in order to form stable loop-loop WC pairingforming a stem which is orthogonal to the main library axis. In otherembodiments, two loops in the aptamer together form an orthogonal stem.In yet other embodiments, the loops in the aptamer contain basesimplanted in order to form stable Hoogsteen pairing with an existingstem along the main library axis. In other embodiments, the loops in theaptamer can form Hoogsteen pairing with any stem in the aptamer.

In some embodiments of the formulae above, the aptamer sequence furthercontains one or more multimer-forming domains.

In some embodiments of the formulae above, the aptamer sequence furthercontains one or more spacers (e.g., of about 1 to 20 bases in length or1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20bases in length).

The aptamers of the present disclosure can be prepared in a variety ofways. In one embodiment, the aptamers are prepared through chemicalsynthesis. In another embodiment, the aptamers are prepared throughenzymatic synthesis. In one embodiment, the enzymatic synthesis can becarried out using any enzyme that can add nucleotides to elongate aprimer, with or without template. In some embodiments, the aptamers areprepared by assembling together k-mers (e.g., k≥2 bases).

In some embodiments, the aptamers of the present disclosure may containany combination of DNA, RNA, and their natural and/or synthetic analogs.In one embodiment, the aptamer comprises DNA. In one embodiment, theaptamer comprises RNA.

In other embodiments, the aptamers of the present disclosure may containany modification on the 5′ end, 3′ end, or internally. Modifications ofthe aptamers include, but are not limited to, spacers, phosphorylation,linkers, conjugation chemistries, fluorophores, quenchers,photoreactive, and modified bases (e.g., LNA, PNA, UNA, PS, methylation,2-O-methyl, halogenated, superbases, iso-dN, inverted bases, L-ribose,other sugars as backbone, etc.).

In some embodiments, the aptamers of the present disclosure may beconjugated to external, non-nucleic acid molecules on the 5′ end, 3′end, or internally. Non-limiting examples of non-nucleic acid moleculesinclude, but are not limited to. amino acids, peptides, proteins, smallmolecule drugs, mono- and polysaccharides, lipids, antibodies andantibody fragments, or a combination thereof.

The aptamers of the present disclosure may contain any domain which hasa biological function. Non-limiting examples of biological functions ofthe aptamers described herein include, but are not limited to, acting astemplates for RNA transcription, binding to, recognizing, and/ormodulating the activity of proteins, binding to transcription factors,specialized nucleic acid structure (e.g., Z-DNA, H-DNA, G-quad, etc.),and acting as an enzymatic substrate for restriction enzymes, specificexo- and endonucleases, recombination sites, editing sites, or siRNA. Inone embodiment, the aptamers modulate the activity of at least oneprotein. In another embodiment, the aptamers inhibit the activity of atleast one protein. In yet another embodiment, the aptamers inhibit theactivity of at least one protein

In other embodiments, the aptamers of the present disclosure may containany domain for integration into a nucleic acid nanostructure built byany one of several known methods (Shih et al, Nature 427:618-621 (2004);Rothemund, Nature 440:297-302 (2006); Zheng et al, Nature 461:74-77(2009); Dietz et al, Science 325:725-730 (2009); Wei et al, Nature485:623-626 (2012); Ke et al, Science 338:1177-1183 (2012); Douglas etal, Science 335:831-834 (2012), each of which are hereby incorporated byreference). In yet other embodiments, the aptamers of the presentdisclosure may contain any domain that serves a function in molecularlogic and computation (Seelig et al, Science 314:1585-1588 (2006);Macdonald et al, Nano Lett 6:2598-2603 (2006); Qian et al, Nature475:368-372 (2011); Douglas et al, Science 335:831-834 (2012); Amir etal, Nat Nanotechnol 9:353-357 (2014), each of which is herebyincorporated by reference).

In some embodiments, the aptamers of the present disclosure undergo oneor more cycles of negative selection versus a target (e.g., eukaryoticor prokaryotic cell, virus or viral particle, molecule, tissue, or wholeorganism, in-vivo or ex-vivo). In other embodiments, the aptamers of thepresent disclosure undergo one or more cycles of positive selectionversus a target (e.g., eukaryotic or prokaryotic cell, virus or viralparticle, molecule, tissue, or whole organism, in-vivo or ex-vivo).

The aptamers of the present disclosure can be in solution or attached toa solid phase (e.g., surface, particles, resin, matrix, etc.). In someembodiments, the aptamer is attached to a surface. In one embodiment,the surface is a flow cell surface.

In some embodiments, the aptamers of the present disclosure aresynthesized in an aptamer library. The aptamer library of the presentdisclosure can be prepared in a variety of ways. In one embodiment, theaptamer library is prepared through chemical synthesis. In anotherembodiment, the aptamer library is prepared through enzymatic synthesis.In one embodiment, the enzymatic synthesis can be carried out using anyenzyme that can add nucleotides to elongate a primer, with or withouttemplate.

In some embodiments, the aptamers synthesized in an aptamer library maycontain any combination of DNA, RNA, and their natural and/or syntheticanalogs. In one embodiment, the aptamers synthesized in an aptamerlibrary comprise DNA. In one embodiment, the aptamers synthesized in anaptamer library comprise RNA.

In some embodiments, the aptamers synthesized in an aptamer library area nucleic acid (e.g., DNA, RNA, natural or synthetic bases, baseanalogs, or a combination thereof) collection of 10^(K) species (K≥2),with Z copies per species (1≤Z≤K−1).

In other embodiments, the aptamers synthesized in an aptamer library ofthe present disclosure may contain any modification on the 5′ end, 3′end, or internally. Modifications of the aptamers include, but are notlimited to, spacers, phosphorylation, linkers, conjugation chemistries,fluorophores, quenchers, photoreactive modifications, and modified bases(e.g., LNA, PNA, UNA, PS, methylation, 2-O-methyl, halogenated,superbases, iso-dN, inverted bases, L-ribose, other sugars as backbone).

In some embodiments, the aptamers synthesized in an aptamer library maybe conjugated to external, non-nucleic acid molecules on the 5′ end, 3′end, or internally. Non-limiting examples of non-nucleic acid moleculesinclude, but are not limited to. amino acids, peptides, proteins, smallmolecule drugs, mono- and polysaccharides, lipids, antibodies andantibody fragments, or a combination thereof.

The aptamers synthesized in an aptamer library may contain any domainwhich has a biological function. Non-limiting examples of biologicalfunctions of the aptamers described herein include, but are not limitedto, acting as templates for RNA transcription, binding to, recognizing,and/or modulating the activity of proteins, binding to transcriptionfactors, specialized nucleic acid structure (e.g., Z-DNA, H-DNA, G-quad,etc.), acting as an enzymatic substrate for restriction enzymes,specific exo- and endonucleases, recombination sites, editing sites, orsiRNA. In one embodiment, the aptamers synthesized in an aptamer librarymodulate the activity of at least one protein. In another embodiment,the aptamers synthesized in an aptamer library inhibit the activity ofat least one protein. In yet another embodiment, the aptamerssynthesized in an aptamer library inhibit the activity of at least oneprotein

In other embodiments, the aptamers synthesized in an aptamer library maycontain any domain for integration into a nucleic acid nanostructurebuilt by one of several known methods (Shih et al, Nature 427:618-621(2004); Rothemund, Nature 440:297-302 (2006); Zheng et al, Nature461:74-77 (2009); Dietz et al, Science 325:725-730 (2009); Wei et al,Nature 485:623-626 (2012); Ke et al, Science 338:1177-1183 (2012);Douglas et al, Science 335:831-834 (2012), each of which are herebyincorporated by reference). In yet other embodiments, the aptamers ofthe present disclosure may contain any domain that serves a function inmolecular logic and computation (Seelig et al, Science 314:1585-1588(2006); Macdonald et al, Nano Lett 6:2598-2603 (2006); Qian et al,Nature 475:368-372 (2011); Douglas et al, Science 335:831-834 (2012);Amir et al, Nat Nanotechnol 9:353-357 (2014), each of which is herebyincorporated by reference)

In some embodiments, the aptamers synthesized in an aptamer libraryundergo one or more cycles of negative selection versus a target (e.g.,eukaryotic or prokaryotic cell, virus or viral particle, molecule,tissue, or whole organism, in-vivo or ex-vivo). In other embodiments,the aptamers of the present disclosure undergo one or more cycles ofpositive selection versus a target (e.g., eukaryotic or prokaryoticcell, virus or viral particle, molecule, tissue, or whole organism,in-vivo or ex-vivo).

The aptamers synthesized in an aptamer library can be in solution orattached to a solid phase (e.g., surface, particles, resin, matrix,etc.). In some embodiments, the aptamers synthesized in an aptamerlibrary are attached to a surface. In one embodiment, the surface is aflow cell surface.

Immobilized Aptamer Clusters

In certain aspects, provided herein are methods for identifying aptamersthat bind to and/or modulate a target by flowing a sample comprising thetarget across a plurality of aptamer clusters (e.g., clusters ofaptamers from the aptamer libraries provided herein) immobilized on asurface. In certain embodiments the surface can be any solid support. Insome embodiments, the surface is the surface of a flow cell. In someembodiments, the surface is a slide or chip (e.g., the surface of a genechip). In some embodiments, the surface is a bead (e.g., a paramagneticbead).

In certain embodiments, any method known in the art can be used togenerate the immobilized aptamer clusters on the surface. In someembodiments, the aptamer clusters are printed directly onto the surface.For example, in some embodiments, the aptamer clusters are printed withfine-pointed pins onto glass slides, printed using photolithography,printed using ink-jet printing, or printed by electrochemistry onmicroelectrode arrays. In some embodiments, at least about 10², 10³,10⁴, 10⁵, 10⁶ or 10⁷ distinct aptamer clusters are printed onto thesurface. In some embodiments, each aptamer cluster comprises at leastabout 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250,300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000,5000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000,90,000 or 100,000 identical aptamer molecules.

Advantageously, direct printing of microarrays allows for aptamers ofknown sequence to be specifically immobilized at a predeterminedposition on the surface, so subsequent sequencing may be unnecessary.

In certain embodiments, the surface-immobilized aptamer clusters aregenerated by first immobilizing aptamers (e.g., from an aptamer librarydisclosed herein) onto the surface (e.g., wherein the position at whicheach aptamer is immobilized is random). In some embodiments, at leastabout 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ or 10¹⁰ distinct aptamersare immobilized onto the surface. Following aptamer immobilization, alocalized amplification process (e.g., bridge amplification or rollingcircle amplification), is then performed to generate clusters of copiesof each immobilized aptamer positioned proximal to the immobilizationsite of the original immobilized aptamer. In certain embodiments (e.g.,embodiments in which rolling circle amplification is performed) theaptamer cluster is housed in a nano-pit or pore on the surface ratherthan being directly immobilized on the surface. In some embodiments,amplification results in each aptamer cluster comprising at least about10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300,350, 400, 450, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000,10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000or 100,000 identical aptamer molecules. In certain embodiments, theaptamer clusters are then sequenced (e.g., by Illumina sequencing orPolonator sequencing) in order to associate the sequence of each aptamercluster with its position on the surface. If present, complementarystrands can be stripped from the aptamer cluster by washing the surfaceunder conditions not amenable to strand hybridization (e.g., due to saltconcentration and/or temperature) in order to generate clusters ofsingle-stranded aptamers. The surface (e.g., flow cell) is then readyfor use in an aptamer identification method provided herein. In someembodiments, the immobilized aptamer clusters are prepared and/orsequenced on one instrument, and then transferred to a separateinstrument for aptamer identification. In other embodiments, the aptamerclusters are prepared and/or sequenced on the same instrument as is usedfor aptamer identification.

In some embodiments of the methods above, the aptamers or aptamerclusters (e.g., from the aptamer library) comprise an adapter that willbring the aptamers to surface height (e.g., in cases where the surfaceis not flat, such as in flow cells that include pores). In oneembodiment, the aptamers or aptamer clusters are immobilized insidepores on a flow cell surface and adapters are used to bind the aptamerto the surface in order to bring the aptamers to surface height. In someembodiments, the adapter is a nucleic acid adapter (e.g., a sequence ofat least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95 or 100 bases in length). In some embodiments, a sequencecomplementary to the adapter sequence is hybridized to the adapter priorto aptamer screening. In some embodiments, the adapter is a chemicaladapter (e.g., a polymer connecting the aptamer to the surface).

Aptamer Library Screening

In certain aspects, provided herein are methods for identifying one ormore aptamers that specifically bind to and/or modulate a target, themethod generally comprising: (i) contacting a plurality of aptamerclusters immobilized on a surface with the target; and (ii) identifyingthe immobilized aptamer clusters that specifically bind to and/ormodulate the target. Because the sequence of each aptamer cluster isassociated with a specific position on the surface (e.g., determinedaccording to the methods provided herein), the sequence of the aptamerresponsible for the binding/modulation is identified and the position atwhich the target is bound and/or modulated can be determined.

In some embodiments, the target is labeled with and/or comprises adetectable label. The target can be detectably labeled directly (e.g.,through a direct chemical linker) or indirectly (e.g., using adetectably labeled target-specific antibody). In embodiments in whichthe target is a cell, it can be labeled by incubating the target cellwith the detectable label under conditions such that the detectablelabel is internalized by the cell. In some embodiments, the target isdetectably labeled before performing the aptamer screening methodsdescribed herein. In some embodiments, the target is labeled during theperformance of the aptamer screening methods provided herein. In someembodiments, the target is labeled after is it is bound to an aptamercluster (e.g., by contacting the bound target with a detectably labeledantibody). In some embodiments, any detectable label can be used.Examples of detectable labels include, but are not limited to,fluorescent moieties, radioactive moieties, paramagnetic moieties,luminescent moieties and/or colorimetric moieties. In some embodiments,the targets described herein are linked to, comprise and/or are bound bya fluorescent moiety. Examples of fluorescent moieties include, but arenot limited to, Allophycocyanin, Fluorescein, Phycoerythrin,Peridinin-chlorophyll protein complex, Alexa Fluor 350, Alexa Fluor 405,Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 514, Alexa Fluor 532,Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660,Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790,EGFP, mPlum, mCherry, mOrange, mKO, EYFP, mCitrine, Venus, YPet,Emerald, Cerulean and CyPet.

The target can be a non-molecular or a supramolecular target.Non-limiting examples of targets to which the aptamers of the presentdisclosure can bind to and/or modulate include, but are not limited to,cells, bacteria, fungi, archaea, protozoa, viruses, virion particles,synthetic and naturally-occurring microscopic particles, and liposomes.In some embodiments, the target introduced into the flow cell islive/native. In other embodiments, the target introduced into the flowcell is fixed in any solution.

In some embodiments, the target is a cell. In some embodiments, the cellis a prokaryotic cell. In some embodiments, the cell is a bacterialcell. non-limiting examples of bacteria include Aspergillus, Brugia,Candida, Chlamydia, Coccidia, Cryptococcus, Dirofilaria, Gonococcus,Histoplasma, Klebsiella, Legionella, Leishmania, Meningococci,Mycobacterium, Mycoplasma, Paramecium, Pertussis, Plasmodium,Pneumococcus, Pneumocystis, Pseudomonas, Rickettsia, Salmonella,Shigella, Staphylococcus, Streptococcus, Toxoplasma and Vibriocholerae.Exemplary species include Neisseria gonorrhea, Mycobacteriumtuberculosis, Candida albicans, Candida tropicalis, Trichomonasvaginalis, Haemophilus vaginalis, Group B Streptococcus sp., Microplasmahominis, Hemophilus ducreyi, Granuloma inguinale, Lymphopathia venereum,Treponema pallidum, Brucella abortus. Brucella melitensis, Brucellasuis, Brucella canis, Campylobacter fetus, Campylobacter fetusintestinalis, Leptospira pomona, Listeria monocytogenes, Brucella ovis,Chlamydia psittaci, Trichomonas foetus, Toxoplasma gondii, Escherichiacoli, Actinobacillus equuli, Salmonella abortus ovis, Salmonella abortusequi, Pseudomonas aeruginosa, Corynebacterium equi, Corynebacteriumpyogenes, Actinobaccilus seminis, Mycoplasma bovigenitalium, Aspergillusfumigatus, Absidia ramosa, Trypanosoma equiperdum, Babesia caballi,Clostridium tetani, and Clostridium botulinum. In some embodiments, thecell is a eukaryotic cell. In some embodiments, the cell is an animalcell (e.g., a mammalian cell). In some embodiments, the cell is a humancell. In some embodiments, the cell is from a non-human animal, such asa mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer,sheep, goat, llama, chicken, cat, dog, ferret, or primate (e.g.,marmoset, rhesus monkey). In some embodiments, the cell is a parasitecell (e.g., a malaria cell, a leishmanias cell, a cryptosporidium cellor an amoeba cell). In some embodiments, the cell is a fungal cell, suchas, e.g., Paracoccidioides brasiliensis.

In some embodiments, the cell is a cancer cell (e.g., a human cancercell). In some embodiments, the cell is from any cancerous orpre-cancerous tumor. Non-limiting examples of cancer cells includecancer cells from the bladder, blood, bone, bone marrow, brain, breast,colon, esophagus, gastrointestine, gum, head, kidney, liver, lung,nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, oruterus. In addition, the cancer may specifically be of the followinghistological type, though it is not limited to these: neoplasm,malignant, carcinoma, carcinoma, undifferentiated, giant and spindlecell carcinoma, small cell carcinoma, papillary carcinoma, squamous cellcarcinoma, lymphoepithelial carcinoma, basal cell carcinoma, pilomatrixcarcinoma, transitional cell carcinoma, papillary transitional cellcarcinoma, adenocarcinoma, gastrinoma, malignant, cholangiocarcinoma,hepatocellular carcinoma, combined hepatocellular carcinoma andcholangiocarcinoma, trabecular adenocarcinoma, adenoid cystic carcinoma,adenocarcinoma in adenomatous polyp, adenocarcinoma, familial polyposiscoli, solid carcinoma, carcinoid tumor, malignant, branchiolo-alveolaradenocarcinoma, papillary adenocarcinoma, chromophobe carcinoma,acidophil carcinoma, oxyphilic adenocarcinoma, basophil carcinoma, clearcell adenocarcinoma, granular cell carcinoma, follicular adenocarcinoma,papillary and follicular adenocarcinoma, nonencapsulating sclerosingcarcinoma, adrenal cortical carcinoma, endometroid carcinoma, skinappendage carcinoma, apocrine adenocarcinoma, sebaceous adenocarcinoma,ceruminous adenocarcinoma, mucoepidermoid carcinoma, cystadenocarcinoma,papillary cystadenocarcinoma, papillary serous cystadenocarcinoma,mucinous cystadenocarcinoma, mucinous adenocarcinoma, signet ring cellcarcinoma, infiltrating duct carcinoma, medullary carcinoma, lobularcarcinoma, inflammatory carcinoma, paget's disease, mammary, acinar cellcarcinoma, adenosquamous carcinoma, adenocarcinoma w/squamousmetaplasia, thymoma, malignant, ovarian stromal tumor, malignant,thecoma, malignant, granulosa cell tumor, malignant, and roblastoma,malignant, sertoli cell carcinoma, leydig cell tumor, malignant, lipidcell tumor, malignant, paraganglioma, malignant, extra-mammaryparaganglioma, malignant, pheochromocytoma, glomangiosarcoma, malignantmelanoma, amelanotic melanoma, superficial spreading melanoma, maligmelanoma in giant pigmented nevus, epithelioid cell melanoma, bluenevus, malignant, sarcoma, fibrosarcoma, fibrous histiocytoma,malignant, myxosarcoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma,embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, stromal sarcoma,mixed tumor, malignant, mullerian mixed tumor, nephroblastoma,hepatoblastoma, carcinosarcoma, mesenchymoma, malignant, brenner tumor,malignant, phyllodes tumor, malignant, synovial sarcoma, mesothelioma,malignant, dysgerminoma, embryonal carcinoma, teratoma, malignant,struma ovarii, malignant, choriocarcinoma, mesonephroma, malignant,hemangiosarcoma, hemangioendothelioma, malignant, kaposi's sarcoma,hemangiopericytoma, malignant, lymphangiosarcoma, osteosarcoma,juxtacortical osteosarcoma, chondrosarcoma, chondroblastoma, malignant,mesenchymal chondrosarcoma, giant cell tumor of bone, ewing's sarcoma,odontogenic tumor, malignant, ameloblastic odontosarcoma, ameloblastoma,malignant, ameloblastic fibrosarcoma, pinealoma, malignant, chordoma,glioma, malignant, ependymoma, astrocytoma, protoplasmic astrocytoma,fibrillary astrocytoma, astroblastoma, glioblastoma, oligodendroglioma,oligodendroblastoma, primitive neuroectodermal, cerebellar sarcoma,ganglioneuroblastoma, neuroblastoma, retinoblastoma, olfactoryneurogenic tumor, meningioma, malignant, neurofibrosarcoma,neurilemmoma, malignant, granular cell tumor, malignant, malignantlymphoma, Hodgkin's disease, Hodgkin's lymphoma, paragranuloma,malignant lymphoma, small lymphocytic, malignant lymphoma, large cell,diffuse, malignant lymphoma, follicular, mycosis fungoides, otherspecified non-Hodgkin's lymphomas, malignant histiocytosis, multiplemyeloma, mast cell sarcoma, immunoproliferative small intestinaldisease, leukemia, lymphoid leukemia, plasma cell leukemia,erythroleukemia, lymphosarcoma cell leukemia, myeloid leukemia,basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mastcell leukemia, megakaryoblastic leukemia, myeloid sarcoma, and hairycell leukemia.

In some embodiments, the target is a virus. For example, in someembodiments, the virus is HIV, hepatitis A, hepatitis B, hepatitis C,herpes virus (e.g., HSV-1, HSV-2, CMV, HAV-6, VZV, Epstein Barr virus),adenovirus, influenza virus, flavivirus, echovirus, rhinovirus,coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV, dengue virus, papillomavirus, molluscum virus, poliovirus, rabiesvirus, JC virus, or ebola virus

In some embodiments, the target is a protein. In certain embodiments,when protein targets are screened they are immobilized on a bead (e.g.,a detectably labeled bead). In some embodiments, protein targets arelinked to a detectable moiety. Non-limiting examples of target proteinsinclude glycoprotein IIb/IIIa, TNF-α, TNFα receptor, CD52, IL-2Rα, Bcell activating factor, VEGF, CD30, IL-1β, epidermal growth factorreceptor, CD38, RANK ligand, Complement protein C5, CD11a, CD20, CTLA4,PD-1, PD-L1, PD-L2, CD3, alpha-4 integrin, IgE, RSV F protein, IL-6R,ErbB2, IL-12, and IL-23. In some embodiments, the target protein is acancer-associated antigen. Examples of cancer-associated antigensinclude, but are not limited to, adipophilin, AIM-2, ALDH1A1,alpha-actinin-4, alpha-fetoprotein (“AFP”), ALK, ANKRD30A, ARTC1, B-RAF,BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4,BIRC7, CA-125, CA9, CALCA, carcinoembryonic antigen (“CEA”), CALR,CASP-5, CASP-8, CCR5, CD19, CD20, CD22, CD27, CD274, CD30, CD33, CD38,CD40, CD44, CD45, CD52, CD56, CD79, Cdc27, CDK12, CDK4, CDKN2A, CEA,CLEC12A, CLPP, COA-1, CPSF, CSNK1A1, CTAG1, CTAG2, cyclin D1, Cyclin-A1,dek-can fusion protein, DKK1, EFTUD2, EGFR, EGFR variant III, Elongationfactor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA2, EphA3, epithelial tumorantigen (“ETA”), ERBB3, ERBB4, ETV6-AML1 fusion protein, EZH2, FCRL3,FGFS, FLT3-ITD, FN1, FOLR1, G250/MN/CAIX, GAGE-1,2,8, GAGE-3,4,5,6,7,GAS7, glypican-3, GnTV, gp100/Pme117, GPNMB, GM3, GPR112, IL3RA, HAUS3,Hepsin, HER-2/neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDO1,IGF2B3, IL13Ralpha2, Intestinal carboxyl esterase, K-ras, Kallikrein 4,KIF20A, KIT, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, KRAS,LAGE-1, LDLR-fucosyltransferaseAS fusion protein, Lengsin, LGR5, LMP2,M-CSF, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6,MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN,MC1R, MCSP, mdm-2, ME1, Melan-A/MART-1, Meloe, Midkine, MMP-2, MMP-7,MUC1, MUC2, MUC3, MUC4, MUC5, MUC5AC, MUC16, mucin, MUM-1, MUM-2, MUM-3,Myosin, Myosin class I, N-raw, NA88-A, neo-PAP, NFYC, NY-BR-1,NY-ESO-1/LAGE-2, OA1, OGT, OS-9, OX40, P polypeptide, p53, PAP, PAX3,PAX5, PBF, PLAC1, PMEL, pml-RARalpha fusion protein, polymorphicepithelial mucin (“PEM”), PPP1R3B, PRAME, PRDX5, PRLR, PSA, PSMA, PTPRK,RAB38/NY-MEL-1, RAGE-1, RBAF600, RET, RGS5, RhoC, RNF43, ROR1, RU2AS,SAGE, SART1, SART3, secernin 1, SIRT2, SLAMF7, SLC39A6, SNRPD1, SOX10,Sp17, SPA17, SSX-2, SSX-4, STEAP1, STEAP2, survivin, SYT-SSX1 or -SSX2fusion protein, TAG-1, TAG-2, Telomerase, TERT, TGF-betaRII,Thompson-nouvelle antigen, TMPRSS2, TNFRSF17, TPBG, TRAG-3,Triosephosphate isomerase, TRP-1/gp75, TRP-2, TRP2-INT2, tyrosinase,tyrosinase (“TYR”), UPK3A, VEGF, VTCN1, WT1, and XAGE-1b/GAGED2a. Insome embodiments, the target protein is a neo-antigen.

In some embodiments, the property of the cell that is modulated is cellviability, cell proliferation, gene expression, cellular morphology,cellular activation, phosphorylation, calcium mobilization,degranulation, cellular migration, and/or cellular differentiation. Incertain embodiments, the target is linked to, bound by or comprises adetectable label that allows for the detection of a biological orchemical effect on the target. In some embodiments, the detectable labelis a fluorescent dye. Non-limiting examples of fluorescent dyes include,but are not limited to, a calcium sensitive dye, a cell tracer dye, alipophilic dye, a cell proliferation dye, a cell cycle dye, a metabolitesensitive dye, a pH sensitive dye, a membrane potential sensitive dye, amitochondrial membrane potential sensitive dye, and a redox potentialdye. In one embodiment, the target is labeled with a calcium sensitivedye, a cell tracer dye, a lipophilic dye, a cell proliferation dye, acell cycle dye, a metabolite sensitive dye, a pH sensitive dye, amembrane potential sensitive dye, a mitochondrial membrane potentialsensitive dye, or a redox potential dye.

In certain embodiments, the target is labeled with an activationassociated marker, an oxidative stress reporter, an angiogenesis marker,an apoptosis marker, an autophagy marker, a cell viability marker, or amarker for ion concentrations. In yet another embodiment, the target islabeled with an activation associated marker, an oxidative stressreporter, an angiogenesis marker, an apoptosis marker, an autophagymarker, a cell viability marker, or a marker for ion concentrationsprior to exposure of aptamers to the target.

In some embodiments, the target is labeled after to exposure of aptamersto the target. In one embodiment, the target is labeled withfluorescently-labeled antibodies, annexin V, antibody fragments andartificial antibody-based constructs, fusion proteins, sugars, orlectins. In another embodiment, the target is labeled withfluorescently-labeled antibodies, annexin V, antibody fragments andartificial antibody-based constructs, fusion proteins, sugars, orlectins after exposure of aptamers to the target.

In some embodiments, the target cell is labeled with a fluorescent dye.Non-limiting examples of fluorescent dyes include, but are not limitedto, a calcium sensitive dye, a cell tracer dye, a lipophilic dye, a cellproliferation dye, a cell cycle dye, a metabolite sensitive dye, a pHsensitive dye, a membrane potential sensitive dye, a mitochondrialmembrane potential sensitive dye, and a redox potential dye.

In some embodiments, the target cell is labeled with a calcium sensitivedye, a cell tracer dye, a lipophilic dye, a cell proliferation dye, acell cycle dye, a metabolite sensitive dye, a pH sensitive dye, amembrane potential sensitive dye, a mitochondrial membrane potentialsensitive dye, or a redox potential dye. In certain embodiments, thetarget cell is labeled with an activation associated marker, anoxidative stress reporter, an angiogenesis marker, an apoptosis marker,an autophagy marker, a cell viability marker, or a marker for ionconcentrations. In yet another embodiment, target cell is labeled withan activation associated marker, an oxidative stress reporter, anangiogenesis marker, an apoptosis marker, an autophagy marker, a cellviability marker, or a marker for ion concentrations prior to exposureof aptamers to the cell. In some embodiments, the target cell is labeledafter to exposure of aptamers to the target. In one embodiment, thetarget cell is labeled with a fluorescently-labeled antibody orantigen-binding fragment thereof, annexin V, a fluorescently-labeledfusion protein, a fluorescently-labeled sugar, or fluorescently labeledlectin. In one embodiment, the target cell is labeled with afluorescently-labeled antibody or antigen-binding fragment thereof,annexin V, a fluorescently-labeled fusion protein, afluorescently-labeled sugar, or fluorescently labeled lectin afterexposure of aptamers to the cell.

The position of the detectable marker on the surface can be determinedusing any method known in the art, including, for example, fluorescentmicroscopy.

FIG. 1 provides an exemplary workflow illustrating certain embodimentsof the methods provided herein. The workflow begins with an initialaptamer library (e.g., an aptamer library provided herein) chosen andprepared as though for Illumina sequencing. The library can be, forexample, newly synthesized, or an output of a previous selectionprocess. This process can involve one or more positive selection cycles,one or more negative selection cycles, or both, in either combinationand sequence.

The prepared library is mounted on adapters on an Illumina flow cell.Bridge PCR amplification turns each single sequence from the initiallibrary into a cluster of about 100,000 copies of the same sequence. Thelibrary is then Illumina-sequenced. This process produces a map linkingeach sequence from the library to a specific set of coordinates on theflow cell surface.

The complementary strands to those from the library, added in theprocess of sequencing by synthesis, are stripped by any one of a numberof methods (e.g., detergents, denaturing agents, etc.). Theoligonucleotide strands complementary to the Illumina adapter and to thePCR primers are then pumped into the flow cell, leaving only the aptamerregion single-stranded. When RNA aptamers are being synthesized as partof the library, transcription is initiated and halted by any one of anumber of methods (e.g., Ter-bound Tus protein, or biotin-boundstreptavidin protein).

The flow cell temperature is raised and then cooled, in order to allowall oligonucleotides on the surface to assume their proper 3D structure,folding according to a folding protocol. In this state, the oligolibrary is folded and ready to engage targets.

The solution comprising the targets is run into the flow cell using theinstrument's hardware. The targets can be labeled prior to introductioninto the flow cell/instrument with a fluorescent dye, for the purpose ofreporting a biological or chemical effect on the target. The targets areincubated for a certain amount of time to allow the effect to takeplace. Fluorescent dyes or markers for reporting the biological orchemical effect (e.g., cell activation, apoptosis, etc.) can then bepumped into the flow cell. (See FIG. 1)

Affected targets (hits) are recognized by image analysis, andcorresponding sequences are analyzed. Extracted sequences aresynthesized and tested separately for binding and function.

EXAMPLES Example 1—Preparation of Aptamer Library

Aptamer libraries were prepared using an Illumina high throughputsequencing platform sample preparation kits which included theattachment of an adapter DNA sequence on the flanks of the samplesequence to complement strands already attached to the surface of theflow cell. The prepared library was mounted onto adapters on the surfaceof an Illumina flow cell.

For the preparation of the aptamer libraries, a two-step “tail” PCRprocess was used to attach the adapters. The PCR reaction mix containedthe following components shown in Table 1:

TABLE 1 Component Amount in μl Herculase II fusion DNA polymerase 0.5buffer 10 Dntp (10 mM each) 1.25 Forward tail primer 1 Reverse tailprimer 1 upw 35.25 sample 1

The primers were set in a way that adapters would have a specificorientation with respect to the sample sequence. This was done to holdthe forward aptamer sequence in the clusters in a single read run.

The sequence of the primers used in 1st PCR reaction:

TruSeq p7 side stab forward primer [SEQ ID NO: 1]GTCACATCTCGTATGCCG TCTTCTGCTTG ATCCAGAGTGACGCAGCA; andTruSeq p5 side stab reverse primer [SEQ ID NO: 2]CTCTTTCCCTACACGACG CTCTTCCGATCT ACTAAGCCACCGTGTCCA

The PCR program used for the first reaction is shown herein below inTable 2:

TABLE 2 Step Temperature Time (seconds) 1 95 180 2 95 30 3 56 10 4 72 105 Return to step 2 × 3  6 95 30 7 85 10 8 72 10 9 Return to step 6 × 1010 4 Forever

The product of first PCR reaction (PCR 1) is the input for the 2nd PCRreaction.

The sequence of the primers used in the 2nd PCR reaction:

TruSeq p7 side start [SEQ ID NO: 3] GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCTCGTATGCCG; and TruSeq p5 side start [SEQ ID NO: 4]AATGATACGGCGACCACCGAGATCTACACACAC TCTTTCCCTACACGACG.

The PCR program used for the second reaction is shown herein below inTable 3:

TABLE 3 Step Temperature time 1 95 30 2 67 10 3 72 10 4 95 30 5 65 10 672 10 7 95 30 8 63 10 9 72 10 10 95 30 11 62 10 12 72 10 13 95 30 14 8710 15 72 10 16 Return to step 13 × 1 17 95 30 18 85 10 19 72 10 20Return to step 17 × 7 21 4 Forever

Completed libraries underwent quality control which included qbit checkfor concentration and tapstation/fragment analyzer to check for librarysize and byproducts. Cluster generation and sequencing was carried outaccording to the sequencing platform and Illumina protocols. After thesequencing process, denaturation provides the clusters in a singlestrand form. Adapters and primers are then blocked and aptamers willfold to their 3d conformation in their folding buffer.

Generation and Sequencing of Clusters

Bridge PCR amplification was used to turn each single sequence from theinitial library into a cluster of about 100,000 copies of the samesequence. The cluster library was then Illumina-sequenced. This processproduced a map linking each sequence from the library to a specific setof coordinates on the flow cell surface.

The complementary strands to those from the library, added in theprocess of sequencing by synthesis, were stripped and oligonucleotidestrands complementary to the Illumina adapter and to the PCR primerswere pumped to the flow cell, leaving only the aptamer regionsingle-stranded. In case of RNA aptamers, transcription was initiatedand halted by any one of a number of methods (e.g., Ter-bound Tusprotein, or biotin-bound streptavidin protein).

The flow cell temperature was raised and then cooled, to allow alloligonucleotides on the surface of the flow cell to assume their proper3D conformation in the appropriate folding buffer. For example, onefolding buffer recipe used (cellselex paper) included 1 liter PBS, 5 mlof 1M MgCl₂, and 4.5 g glucose

Target Introduction

Target (e.g., cells, bacteria, particles, viruses, proteins, etc.) wereintroduced into the system in the desired binding buffer according tothe environment they would be used in (e.g., human serum, PBS, lb) usingthe machine's hardware. One option for a general binding buffer recipeis (cellselsex paper): 1 liter PBS, 5 ml 1M MgCl₂, 4.5 g glucose, 100 mgtRNA, and 1 g BSA. Targets were labeled prior to or after introductioninto the flow cell/machine and incubated for a certain amount of time tolet effect take place.

Targets can be labeled using different fluorophore that will fit theplatforms excitation source and emission filters. Labeling can be donethrough any possible docking site available on the target. Examples oflabeling agents include, but are not limited to, DiI, anti HLA+seconderyDylight 650, anti HLA PE-Cy5, and Dylight 650.

For the screening of functional aptamers, fluorescent reporters can beused to visualize the effect. For example, introduction of 7AAD to theflow cell can be used to label the targets to screen for cell death, orannexin V fluorophore conjugate can be used to label the targets toscreen for apoptosis. The reporter agent, its concentration, time ofincubation and specific recipe protocol should be adjusted in accordancewith the specific effect screening for.

Representative method for sequencing initial library followed by targetcell introduction and acquisition of functional oligonucleotide clusters80 μl of “Incorporation Mix Buffer” is pumped into the flow cell at arate of 250 μl/min. The temperature is then set temperature to 55° C. 60μl of “Incorporation Mix” is pumped to the flow cell at a rate of 250μl/min and after 80 seconds 10 μl of “Incorporation Mix” is pumped tothe flow cell at a rate of 250 μl/min. After 211 seconds, thetemperature is set to 22° C. and 60 μl of “Incorporation Mix Buffer” ispumped to the flow cell at a rate of 250 μl/min. 75 μl of “Scan Mix” isthen pumped into to the flow cell at a rate of 250 μl/min.

The method then calibrates to focus to the plane of the clusters andalign microscope and flow cell planes. 100 μl of “Incorporation MixBuffer” is pumped into to the flow cell at a rate of 250 μl/min. Theincorporation steps above are repeated 99 times.

The temperature control is turned off and 125 μl of “Cleavage Buffer” ispumped into the flow cell at a rate of 250 μl/min. The temperature isthen set to 55° C. and 75 μl of “Cleavage Mix” pumped into the to theflow cell at a rate of 250 μl/min. After 80 seconds, 25 μl of “CleavageMix” is pumped into the flow cell at a rate of 250 μl/min.

After an addition 80 seconds, 25 μl of “Cleavage Mix” is pumped into theflow cell at a rate of 250 μl/min. After 80 seconds, the temperature isset to 22° C. The temperature control is then turned off and 60 μl of“Incorporation Mix Buffer” is pumped into the flow cell at a rate of 250μl/min. The volume remaining in each water tube is then checked toverify proper delivery.

Denaturation then takes place followed by capping. For the denaturationsteps, the temperature is then set to 20° C. for 120 seconds. 75 μl of“Wash Buffer” is pumped into the flow cell at a rate of 60 μl/min,followed by 75 μl of “Denaturation Solution” at a rate of 60 μl/min and75 μl of “Wash Buffer” at a rate of 60 μl/min.

For the capping steps, 75 μl of “Wash Buffer” is pumped into the flowcell at a rate of 60 μl/min and the temperature is set to 85° C. for 120seconds. 80 μl of “5′ Cap” is then pumped into the flow cell at a rateof 80 μl/min and the temperature is set to 85° C. for 30 seconds. 10 μlof “5′ Cap” is pumped into the flow cell at a rate of 13 μl/min and thetemperature is set to 85° C. for 60 seconds. 10 μl of “5′ Cap” is pumpedinto the flow cell at a rate of 13 μl/min and the temperature is set to85° C. for 90 seconds. 10 μl of “5′ Cap” is pumped into to the flow cellat a rate of 13 μl/min and the temperature is set to 85° C. for 120seconds. 10 μl of “5′ Cap” is pumped into the flow cell at a rate of 13μl/min and the temperature is set to 85° C. for 150 seconds. 10 μl of“5′ Cap” is pumped into the flow cell at a rate of 13 μl/min and thetemperature is set to 85° C. for 180 seconds. 10 μl of “5′ Cap” ispumped into the flow cell at a rate of 13 μl/min and the temperature isset to 85° C. for 210 seconds. 10 μl of “5′ Cap” is pumped into the flowcell at a rate of 13 μl/min and the temperature is set to 85° C. for 240seconds. 10 μl of “5′ Cap” is pumped into the flow cell at a rate of 13μl/min and the temperature is set to 85° C. for 270 seconds. 75 μl of“Wash Buffer” is pumped into the flow cell at a rate of 60 μl/min andthe temperature is set to 85° C. for 120 seconds.

For the 3′ Cap, 80 μl of “3′ Cap” is pumped into the flow cell at a rateof 80 μl/min and the temperature is set to 85° C. for 30 seconds. 10 μlof “3′ Cap” is pumped into the flow cell at a rate of 13 μl/min and thetemperature is set to 85° C. for 60 seconds. 10 μl of “3′ Cap” is pumpedinto the flow cell at a rate of 13 μl/min and the temperature is set to85° C. for 90 seconds. 10 μl of “3′ Cap” is pumped into the flow cell ata rate of 13 μl/min and the temperature is set to 85° C. for 120seconds. 10 μl of “3′ Cap” is pumped into the flow cell at a rate of 13μl/min and the temperature is set to 85° C. for 150 seconds. 10 μl of“3′ Cap” is pumped into the flow cell at a rate of 13 μl/min and thetemperature is set to 85° C. for 180 seconds. 10 μl of “3′ Cap” ispumped into the flow cell at a rate of 13 μl/min and the temperature isset to 85° C. for 210 seconds. 10 μl of “3′ Cap” is pumped into the flowcell at a rate of 13 μl/min and the temperature is set to 85° C. for 240seconds.

10 μl of “3′ Cap” is pumped into the flow cell at a rate of 13 μl/minand the temperature is set to 85° C. for 270 seconds. 75 μl of “WashBuffer” is pumped into the flow cell at a rate of 60 μl/min and thetemperature is set to 0° C. 200 μl of “Folding Buffer (chilled)” ispumped into the flow cell at a rate of 250 μl/min followed by 160 μl of“Folding Buffer (chilled)” at a rate of 40 μl/min and the temperature isset to 0° C. for 600 seconds.

The temperature is raised to 37° C. for 120 seconds. This is followed bya binding step.

For the binding step, 80 μl of “Binding Buffer” is pumped into the flowcell at a rate of 250 μl/min and the temperature is set to 37° C. 80 μlof “Target #1” is pumped into the flow cell at a rate of 100 μl/min andthe temperature is set to 37° C. for 300 seconds. 10 μl of “Target #1”is again pumped into the flow cell at a rate of 13 μl/min and thetemperature is set to 37° C. for 300 seconds. Lastly, 10 μl of “Target#1” is pumped into to the flow cell at a rate of 13 μl/min and thetemperature is set to 37° C. for 2700 seconds.

This is followed by a three consecutive incorporation steps and washsteps to remove unbound target consisting of incorporation, pumping 80μl of “Binding Buffer” into the flow cell at a rate of 13 μl/min,incorporation, pumping 80 μl of “Binding Buffer” into the flow cell at arate of 80 μl/min, incorporation, pumping 80 μl of “Binding Buffer” intothe flow cell at a rate of 200 μl/min and incorporation.

The denaturing, capping, binding, incorporation and washing steps aboveare repeated until sequencing and target introduction is complete.Various targets are then added and binding to and/or activity of theaptamers is evaluated.

FIG. 3 shows a time lapse image of the movement of a Hana cell bound tothe flow cell. The results demonstrate that the cell is actually boundby the sequences attached to the surface itself, rather than the surfaceitself, and is thus free to move but confined to that location.

Example 2—Functional Aptamer Identification

An embodiment of the method provided herein was used to identifyaptamers that induce apoptosis in freshly isolated (12 hr) human triplenegative breast cancer cells. An aptamer library as described herein wasimmobilized on a flow cell and bridge

PCR was performed in an Illumina instrument to generate a clusteredlibrary. During the amplification process the clustered library wassequenced according to the Illumina protocol with the exception that PBSwas used in place of bleaching reagent at the end of the sequencingprocess so that the flow cell was not destroyed following sequencing.The known probes made up 0.1-1% of the library so that the resultingsequencing map could be aligned to later generated fluorescentmicroscope images.

After the final PBS wash, the flow cell was loaded onto a fluorescentmicroscope with temperature control, and clusters were imaged at phaseto view clusters and in the green fluorescence channel to view theapoptosis indicator. The coordinates of the known probe sequences wereused to align the microscope-generated image with the sequence clustermap generated during the sequencing process by the sequencer.

Freshly isolated human breast cancer cells from bone marrow aspiratewere prepared at 10⁶ cells/mL concentration to achieve an average celldensity of ˜1 per 100 micron² on the flow cell chamber floor in PBScontaining 1% albumin and 1 mM Mg²⁺. Target cells were loaded with agreen fluorescent caspase 3/7 activity reporter dye as per themanufacturer's instructions and washed. Cells were pumped into the flowcell using a syringe pump.

The flow cell was imaged at t=0 in Phase and Green channel and thenincubated at 37 degrees and imaged again at 30 minute intervals.Aptamer-induced apoptosis was detected by green fluorescence when thetarget cell was engaged by a functional aptamer. Cells were washed withthe same buffer under increasing pump pressure and further imaged toidentify functional aptamers that had a higher affinity for the targetcells.

The clustered aptamer library analysis was repeated using peripheralblood mononuclear cells as a counter-target in a different lane of theflow-cell using the same sequenced library to identify target-specificaptamer sequences. Computational analysis was performed to translatecoordinates at which target or counter-target cells bound to aptamerclusters and underwent apoptosis to identify aptamer sequences thatpreferentially mediated apoptotic function in the target cells versusthe counter-target cells. More than 1,000 aptamers capable ofspecifically mediating target-selective apoptosis on tumor cells fromthe specific donor were successfully identified from the library.

Example 3—Functional Aptamer Identification in an Illumina Sequencer

An embodiment of the method provided herein is used to identify aptamersthat induce apoptosis in freshly isolated (12 hr) human tumor cells.

An aptamer library as described herein is immobilized on a flow cell andbridge PCR was performed in an Illumina instrument to generate aclustered library. During the amplification process the clusteredlibrary is sequenced according to the Illumina protocol with theexception that PBS was used in place of bleaching reagent at the end ofthe sequencing process so that the flow cell is not destroyed followingsequencing.

After the final PBS wash, the clusters are imaged in the IlluminaInstrument in the green fluorescence channel to view the apoptosisindicator. Freshly isolated human breast cancer cells from bone marrowaspirate are prepared at 10⁶ cells/mL concentration to achieve anaverage cell density of ˜1 per 100 micron² on the flow cell chamberfloor in PBS containing 1% albumin and 1 mM Mg²⁺. Target cells areloaded with a green fluorescent caspase 3/7 activity reporter dye as perthe manufacturer's instructions and washed. Cells are pumped into theflow cell using the Illumina instrument.

An additional sequencing cycle is run during which the flow cell isimaged. The position of target cells within the flow cell is determinedbased on the presence of a cell-specific fluorescent signal.Aptamer-induced apoptosis is detected by the appearance of fluorescentsignal at flow cell coordinates associated with one or more aptamerclusters. Cells appear to the sequencer as “spots” of several clusters.Target cells undergoing apoptosis are identified based the fluorescenceemission of the activity reporter dye.

The clustered aptamer library analysis is repeated using non-tumor cellsas a counter-target in a different lane of the flow-cell using the samesequenced library to identify target-specific aptamer sequences.Computational analysis is performed to translate coordinates at whichtarget or counter-target cells bound to aptamer clusters and underwentapoptosis to identify aptamer sequences that preferentially mediatedapoptotic function in the target cells versus the counter-target cells.Aptamers capable of specifically mediating target-selective apoptosis ontumor cells from the specific donor are identified from the library.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned herein arehereby incorporated by reference in their entirety as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method for identifying one or more aptamersthat mediate a cell function in a target cell, the method comprising:(i) contacting a plurality of surface-immobilized aptamer clusters withthe target cell; and (ii) identifying immobilized aptamer clusters thatmediate the cell function in the target cell.
 2. The method of claim 1,wherein the surface-immobilized aptamer clusters are immobilized onbeads.
 3. The method of claim 2, wherein the beads are paramagneticbead.
 4. The method of claim 2, wherein the beads are detectablylabeled.
 5. The method of claim 1, further comprising the steps of: (a)immobilizing a plurality of aptamers from an aptamer library onsurfaces; and (b) amplifying the plurality of immobilized aptamerslocally on the surfaces to form the plurality of immobilized aptamerclusters.
 6. The method of claim 5, wherein the amplification isconducted via bridge PCR amplification or rolling circle amplification.7. The method of claim 6, wherein the method further comprises removingthe complementary strands from the immobilized aptamer clusters toprovide single stranded immobilized aptamer clusters.
 8. The method ofclaim 1, further comprising an aptamer folding step, wherein the aptamerfolding step comprises raising and then lowering the temperature of thesurface.
 9. The method of claim 1, further comprising an aptamer foldingstep, wherein the aptamer folding step comprises adding a denaturingagent to the surfaces and then removing the denaturing agent from thesurfaces.
 10. The method of claim 1, further comprising the step ofsequencing the aptamer clusters identified in step (ii).
 11. The methodof claim 10, wherein the sequencing is conducted via Illunmia sequencingor Polonator sequencing.
 12. The method of claim 1, wherein at least 10⁸distinct aptamers are immobilized on the one or more surfaces and eachaptamer cluster comprises 10³ to 10⁶ copies of an aptamer.
 13. Themethod of claim 1, wherein the target cell is detectably labeled. 14.The method of claim 1, wherein the target cell is a mammalian cell. 15.The method of claim 14, wherein the mammalian cell is a human cell. 16.The method of claim 15, wherein the human cell in an immune cell. 17.The method of claim 15, wherein the human cell is a cancer cell.
 18. Themethod of claim 15, wherein the cell function is cell death, caspase-3/7activity, proliferation, gene expression, or cytokine expression. 19.The method of claim 18, wherein the cell function is cell death.
 20. Themethod of claim 19, wherein the target cell is labeled with afluorescent reporter of cell death.